Method for increasing productivity of secondary metabolite by conferring drug-resistant mutations

ABSTRACT

This invention relates to a method for increasing the productivity of the secondary metabolite in a micro-organism, for example, the improvement of antibiotic producers by conferring drug-resistant mutations to micro-organisms.

TECHNICAL FIELD

[0001] This invention relates to a method for increasing theproductivity of the secondary metabolite such as antibiotics in amicroorganism by conferring a drug-resistance to the microorganism. Moreparticularly, the present invention relates to the improvement of thesecondary metabolite by conferring combined drug-resistant mutations tomicroorganisms.

TECHNICAL BACKGROUND

[0002] “Secondary metabolites” was originally used by plantphysiologists to classify botanicals (e.g. dyes, fragrances, andmedicinals) with no obvious function in the plants that produced them.It now encompasses a heterogous group of compounds, usually of lowrelative molecular mass, and made mostly but not exclusively byorganisms without a nervous system (i.e., bacteria, fungi, and plants).The notion of secondary metabolism was embrassed by microbiologists inthe 1960s, with attention focusing on antibiotics and other bioactivemicrobial products (Bently, R. et al, Annu. Rev. Microbio. (1999) 53:411-446).

[0003] It is well known that members of the genus Streptomyces produce agreat many antibiotics and other classes of biologically activesecondary metabolites. The genus Streptomyces belongs to the orderActinomycetales. In general, the order Actinomycetales meansActinomycetes. Actinomycetes make over 60% of the known secondarymetabolites that are produced by microorganisms, and amongst them nearly80% are made by members of the genus Streptomyces, with other generatrailing numerically (Kieser, T. et al, Practical Streptomyces Genetics(2000): 10-11).

[0004] Most antibiotics applied in clinical were first isolated from themetabolites of microbes including bacteria, fungi and actinomycetes, inwhich Streptomyces are known to be the most effective producers. Geneticrecombination and manipulation in Streptomyces have been established byD. A. Hopwood and his co-workers (Hopwood et al. Genetic manipulation ofStreptomyces, a laboratory manual, 1985). So far, treatment withmutagens and the screening of resultant clones has been repeated andadopted as a procedure for the improvement of antibiotic-producingStreptomyces and produced good results. However, this method has somedisadvantageous properties such as labor-consuming, time-consuming,costly, non-reproductive and low frequency etc. The current intentionalmethods for improving strains are genetic recombination and manipulationthat represent an important technique in strain improvement such asprotoplast fusion, structural gene amplification, regulatory genes andresistance genes cloning etc. (Ikeda et al. Actinomycetologica 1991,5:86-99). Obviously, these methods require a better knowledge aboutbiochemistry and genetics of antibiotic production.

[0005] As described above, some methods for improving strains have beeninvented and applied in fermentation industry. It is known that aproductivity of actinorhodin in Streptomyces coelicolor can be improvedby conferring streptomycin resistance to it (Protein, Nucleic Acid,Enzyme, vol. 44, No. 13, p 1967-1974 (1999); Kagaku to Seibutsu, vol.37, No. 11, p 731-737 (1999)). However, no report has ever been madeconcerning more effective methods to increase the productivity of thesecondary metabolites.

[0006] An object of the present invention is to provide a method forincreasing the productivity of the secondary metabolite by amicroorganism in a labor-saving, time-saving, high efficient andsemi-rational way and being applicable for the strains without moreknowledge of, for example, antibiotic biochemistry and genetics.

DISCLOSURE OF THE INVENTION

[0007] In order to accomplish the object as described above, theinventors made extensive studies and found the following unexpected newfindings. That is, the actinorhodin productivity in Streptomycescoelicolor can be improved by conferring a resistance against two ormore antibiotics, such as: streptomycin, geneticin, gentamicin andrifampicin. Each antibiotic possesses the ability to increase theproductivity by inducing the respective mutations. Using onestreptomycin-resistant mutant as starting strain, the productivity ofactinorhodin could be further increased by introducing anotherantibiotic-resistant mutation such as: geneticin-resistant,gentamicin-resistant or rifampicin-resistant mutation, which means thatdouble mutations (streptomycin and geneticin, streptomycin andgentamicin, streptomycin and rifampicin combined resistant mutations)could continuously increase productivity of actinorhodin. Finally, byintroducing rifampicin-resistant mutation into the double (streptomycinand gentamicin) mutants, the productivity could be increased further. Itwas confirmed that by introducing combined three antibiotic resistantmutations, the productivity of actinorhodin in Streptomyces coelicolorcould continuously increase in a stepwise way and reach a high producinglevel.

[0008] The present invention was accomplished based on the new findingsdescribed above.

[0009] Thus, the present invention provides:

[0010] (1) A method for increasing a productivity of a secondarymetabolite in a microorganism by conferring a resistance against two ormore antibiotics to said microorganism.

[0011] (2) A method for obtaining a microorganism having an increasedproductivity of a secondary metabolite, which comprises the steps ofconferring a resistance against two or more antibiotics to saidmicroorganism by culturing it in a medium containing the antibiotics,wherein the concentration of said antibiotics is higher than MIC of saidantibiotics against the original microorganism, and isolating colonieswhich can grow in the medium.

[0012] (3) The method as described in (1) or (2) above, wherein saidmicroorganism is a bacterium.

[0013] (4) The method as described in (1) or (2) above, wherein saidmicroorganism belongs to the genus selected from the group consisting ofStreptomyces, genus Bacillus, and genus Pseudomonas.

[0014] (5) The method as described in (1) or (2) above, wherein saidmicroorganism is selected from the group consisting of Streptomycescoelicolor, Streptomyces lividans, Streptomyces antibioticus,Streptomyces chattanoogensis, Bacillus subtilis, Bacillus cereus, andPseudomonas pyrrocinia.

[0015] (6) The method as described in (1) or (2) above, wherein saidantibiotic is selected from the group consisting of ribosomalprotein-attacking antibiotics, ribosomal RNA-attacking antibiotics, andRNA polymerase-attacking antibiotics.

[0016] (7) The method as described in (1) or (2) above, wherein saidantibiotic is selected from the group consisting of streptomycin,geneticin, gentamicin, and rifampicin.

[0017] (8) The method as described in (1) or (2) above, wherein saidsecondary metabolite is selected from the group consisting ofantibiotics, enzymes and physiologically active substances.

[0018] (9) A method for producing a secondary metabolite, whichcomprises the steps of

[0019] culturing in a medium a microorganism having a resistance againstat least two antibiotics and having an increased productivity of thesecondary metabolite in comparison to the original microorganismthereof,

[0020] forming and accumulating a secondary metabolite, and

[0021] recovering the secondary metabolite therefrom.

[0022] (10) The method as described in (9) above, wherein saidmicroorganism is a bacterium.

[0023] (11) The method as described in (9) above, wherein saidmicroorganism belongs to the genus selected from the group consisting ofStreptomyces, genus Bacillus, and genus Pseudomonas.

[0024] (12) The method as described in (9) above, wherein saidmicroorganism is selected from the group consisting of Streptomycescoelicolor, Streptomyces lividans, Streptomyces antibioticus,Streptomyces chattanoogensis, Bacillus subtilis, Bacillus cereus, andPseudomonas pyrrocinia.

[0025] (13) The method as described in (9) above, wherein saidantibiotic is selected from the group consisting of ribosomalprotein-attacking antibiotic, ribosomal RNA-attacking antibiotics, andRNA polymerase-attacking antibiotics.

[0026] (14) The method as described in (9) above, wherein saidantibiotic is selected from the group consisting of streptomycin,geneticin, gentamicin, and rifampicin.

[0027] (15) The method as described in (9) above, wherein said secondarymetabolite is selected from the group consisting of antibiotics, enzymesand physiologically active substances.

[0028] (16) A microorganism having an increased productivity of thesecondary metabolite in comparison to an original strain thereof, whichis produced in accordance with the method of (1) or (2) above.

[0029] (17) The microorganism as described in (16) above, wherein saidmicroorganism is a bacterium.

[0030] (18) The microorganism as described in (16) above, wherein saidmicroorganism belongs to the genus selected from the group consisting ofStreptomyces, genus Bacillus, and genus Pseudomonas.

[0031] (19) The microorganism as described in (16) above, wherein saidmicroorganism is selected from the group consisting of Streptomycescoelicolor, Streptomyces lividans, Streptomyces antibioticus,Streptomyces chattanoogensis, Bacillus subtilis, Bacillus cereus, andPseudomonas pyrrocinia.

[0032] (20) A method for increasing a productivity of a secondarymetabolite in a microorganism by conferring a resistance against asingle antibiotic, excepting streptomycin, to said microorganism.

[0033] As another aspect of the invention, a method is provided forinducing other antibiotic biosynthesis in microorganisms by introducinga single or multi drug-resistant mutation and providing a new approachto find new antibiotics.

BRIEF DESCRIPTION OF THE DRAWINGS

[0034] This invention is illustrated by reference to the followingdrawings:

[0035]FIG. 1 is the graph indicating the strategy for constructingcombined drug-resistant mutants. Three kinds of single mutants anddouble mutants and one kind of triple mutants were constructed as shown.

[0036]FIG. 2 is a photograph indicating the ability to produce aerialmycelia and actinorhodin in Streptomyces coelicolor wild type and mutantstrains. Spores were inoculated on R4 and R3 agar plates, and incubatedat 30° C. for 6 days.

[0037]FIG. 3 is a graph indicating amino acid alterations in theβ-subunits of RNA polymerases in rifampicin-resistant mutants. ClustersI and II represent the previously known resistance-determining regions.Numbering originates from the starting amino acid (Met) of the openreading frame. Mutation positions are indicated by arrows and numbers,and single capital letters denote the altered amino acids found in thisstudy. Shaded parts indicate the substitutions newly found in our study.

[0038]FIG. 4 is a graph indicating comparison of actinorhodin productionbetween media R3 (□) and R4 (▪). Actinorhodin was determined after 6days of incubation.

[0039]FIG. 5 is a graph indicating actinorhodin production in media R4and R3. Symbols: ▪, 1147 (wild type); □, S-1 (str); , SG-1 (str-gen);◯, SGR-1 (str-gen-rif).

[0040]FIG. 6 is a graph indicating the effect of yeast extract, Casaminoacids and KH₂PO₄ on the production of actinorhodin. Strains were grownfor 6 days in R4 medium supplemented with various concentrations ofyeast extract, Casamino acids or KH₂PO₄. Symbols: ▪, 1147 (wild type);□, S-1 (str); , SG-1 (str-gen); ◯, SGR-1 (str-gen-rif).

[0041]FIG. 7 is a graph indicating increased productivity ofactinorhodin in combined resistant mutants. Cultures were grown for 7days in GYM, R3 or R4 liquid medium. The strains S-1, SG-1 and SGR-1were used as single, double and triple mutants, respectively.

[0042]FIG. 8 is a graph indicating the chemical structure ofactinorhodin produced by Streptomyces coelicolor.

[0043]FIG. 9 is a photograph indicating the potentially newantibacterial agents produced in the mutants of certain Streptomycesstrain No. 618824 by introducing drug-resistant mutations.

[0044]FIG. 10 is a graph indicating the structures of four antibioticsused for conferring the resistance, including gentamicin, geneticin,streptomycin and rifampicin.

BEST MODE FOR CARRYING OUT THE INVENTION

[0045] The microorganism used in the present invention is notparticularly limited as long as the productivity of the secondarymetabolite thereof can be increased by making the microorganism to beresistant to two or more antibiotics. It is preferable to use soilmicroorganism that produce agriculturally and/or medically usefulantibiotics, enzymes and biologically active substances. Preferable oneis bacteria. More preferable one is Actinomycetes. Examples of thosebelonging to Actinomycetes may be exemplified as genus Streptomyces,genus Bacillus, genus Pseudomonas, etc. And the most preferable one isgenus Streptomyces. Specific non-limiting illustrative examples of thebacteria include Streptomyces coelicolor, Streptomyces lividans,Streptomyces antibioticus, Streptomyces chattanoogensis, Bacillussubtilis, Bacillus cereus, and Pseudomonas pyrrocinia.

[0046] The microorganism can be isolated from the nature by the knownmethod or available from the culture collections such as ATCC. Themicroorganism may be any of wild strains, mutant strains, cell fusionstrains, transduced strains or recombinant strains constructed by meansof recombinant DNA techniques, and may be any ones which have alreadybeen used in a fermentation industry as producers for agriculturallyand/or medically used antibiotics, enzymes, biologically activesubstances.

[0047] The secondary metabolite is not particularly limited as long asthe productivity thereof can be increased by making the microorganism tobe resistant to one or more antibiotics. Preferable secondarymetabolites include agriculturally and/or medically useful antibiotics(for example: actinomycin from Streptomyces antibioticus 3720,fredericamycin from Streptomyces chattanoogensis ISP5002, FR900493 fromBacillus cereus 2045, and pyrrolnitrin from Pseudomonas pyrrocinia 2327,etc.), enzymes (for example: protease from Bacillus sp., amylase fromBacillus sp., acylase from Streptomyces lavendulae, adenosine deaminasefrom Micrococcus flavus, and demethylase from Streptomyces punipalus,etc.), and biologically active substances (for example: FK506 fromStreptomyces tsukubaensis, WS7739 from Streptomyces phaeofaciensNo.7739, and WS1279 from Streptomyces willmorei No.1279, etc.).

[0048] The antibiotic used in the present invention to introduce anmutation to the microorganism is not particularly limited as long as themicroorganism which is conferred resistant to the antibiotic can producethe secondary metabolite. The preferable antibiotic include those calledribosome-attacking antibiotics including ribosomal protein-attackingantibiotics (e.g., ribosomal S12 protein-attacking antibiotics) andribosomal RNA-attacking antibiotics, and RNA polymerase-attackingantibiotics.

[0049] In order to increase a productivity of a secondary metabolite ina microorganism, it is preferable to confer a resistance against two ormore antibiotics to a microorganism. Particularly, a resistance againsttwo or three antibiotics to a microorganism is more preferable. And themost preferable one is a resistance against three antibiotics to amicroorganism.

[0050] Preferable combination of antibiotics for conferring resistanceis (1) two different aminoglycoside antibiotics, (2) one aminoglycosideantibiotic and one ansamycin antibiotic, and (3) two differentaminoglycoside antibiotics and one ansamycin antibiotic.

[0051] Specific non-limiting examples of the antibiotic includeaminoglycoside class antibiotics such as streptomycin, geneticin andgentamicin and ansamycin class antibiotics such as rifampicin.

[0052] The method for obtaining the antibiotic-resistant mutantmicroorganism from the original microorganism is not particularlylimited. Preferably, an original microorganism is cultured in a mediumcontaining the antibiotic and the antibiotic-resistant spontaneousmutants may be obtained by isolating colonies which can grow on theantibiotic. Alternatively, a microorganism may be subjected to the usualmutation treatment, such as ultraviolet ray irradiation or chemicaltreatment with N-methyl-N′-nitro-N-nitrosoguanidine (NTG) or nitrousacid, and antibiotic-resistant mutants may be obtained by isolatingcolonies which can grow on the antibiotic. In this step, theconcentration of the antibiotic in a medium is preferably controlled tobecome higher than MIC for the original microorganism.

[0053] Preferably, the concentration of antibiotics is two times ormore, more preferably 5 times or more, most preferably 10 times or more,higher than MIC (Minimum Inhibitory Concentration) of the originalmicroorganism. For example, in case that streptomycin is adopted as theantibiotic, the preferable concentration thereof is 5- to 100-timeshigher than MIC against the original microorganism. And in case thatrifampicin is adopted as the antibiotic, the preferable concentrationthereof is 5- to 40-times higher than MIC against the originalmicroorganism. And in case that gentamicin is adopted as the antibiotic,the preferable concentration thereof is 2-times higher than MIC againstthe original microorganism.

[0054] This step may be further repeated to obtain a multi (two ormore)-resistant microorganism which is resistant to two or moreantibiotics.

[0055] Then, a mutant which can produce the intended secondarymetabolite in an increased amount is selected by an appropriate way. Forexample, it may be selected by analytical method (e.g., TLC, HPLC(DAD),LC-MS, optical density etc.), biological assay methods (e.g., assayingactivities of enzyme, such as acylase, demethylase, deaminase, etc.;assaying activities of biologically active substances, such asanti-bacterial, anti-fungal, anti-cancer activities, etc.), and so on.For example, the amount of actinorhodin is determined by measuring theoptical density of supernatants at 633 nm.

[0056] When the multi-resistant microorganism is used, the step ofselecting the strain having an increased productivity of the secondarymetabolite can be carried out after obtaining the multi-resistantmicroorganism or after at least one step of introducing the mutation.

[0057] Using the recombinant DNA techniques, a strain having improvedproductivity of a secondary metabolite of interest can be obtained bytransforming a host with a recombinant plasmid containing a gene for thebiosynthesis of said secondary metabolite.

[0058] In the above-described steps, culturing of the microorganism canbe carried out in accordance by utilizing a generally used culturingmethod.

[0059] The medium may be either a synthetic medium or a natural medium,as long as it contains appropriate amounts of necessary carbon sources,nitrogen sources, inorganic substances, amino acids, vitamins, and/ortrace amounts of nutrient substances. They are shown in various papers,such as Hosoya, Y. et al, Antimicrob. Agents Chemother.(1998)42:2041-2047; Kieser, T. et al, Practical Streptomyces Genetics (2000):406-415; and Media information listed for bacteria in ATCC MediaHandbook.

[0060] Examples of the carbon source include carbohydrates such asglucose, fructose, sucrose, maltose, mannose, glycerol, starch, starchhydrolysate and molasses, polyalcohols and various organic acids such aspyruvic acid, fumaric acid, lactic acid and acetic acid.

[0061] Examples of the nitrogen source include ammonia or variousinorganic and organic ammonium salts such as ammonium chloride, ammoniumsulfate, ammonium carbonate and ammonium acetate, urea and othernitrogen-containing substances, and nitrogen-containing organicsubstances such as peptone, NZ-amine, meat extract, yeast extract, cornsteep liquor, casein hydrolysate and fish meal or a digested productthereof.

[0062] Examples of the inorganic substance include potassiumdihydrogenphosphate, dipotassium hydrogenphosphate, ammonium sulfate,ammonium chloride, magnesium sulfate, sodium chloride, ferrous sulfate,manganese sulfate and calcium carbonate.

[0063] Amino acids and vitamins may be added to the medium as occasiondemands.

[0064] The microorganism may be cultured under aerobic conditions suchas shaking culture or aeration agitation culturing. In general, theculturing is carried out at a temperature of preferably from 20 to 40°C. It is desirable to maintain pH of the medium at around the neutrallevel. The culturing is carried out generally from a period of from 1 to7 days.

[0065] The desired secondary metabolite formed and accumulated in themicroorganism and/or culture medium can be recovered, for example, bythe following method. The cells are removed after completion of theculturing, or the cells are disrupted and the resulting mixture iscentrifuged to remove the disrupted cells. Then, the desired metaboliteis recovered by a known method suitable for recovering the desiredsecondary metabolite, such as a concentration crystallization method, anactivated carbon treatment method and/or an ion exchange resin method.

[0066] The present invention is further described taking Streptomycesstrains as an illustrative example, but the present invention should notbe construed as being limited thereto.

[0067] The strains belonging to the genus Streptomyces are explainedbelow as the examples of the microorganism of the present invention,which can be divided into two groups: the wild type strain anddrug-resistant mutants of Streptomyces coelicolor and non-identifiedStreptomyces sp. isolated from soils which normally do not produceantibiotics. Four kinds of antibiotics named as gentamicin, geneticin,streptomycin and rifampicin are explained below as the examples of thedrug.

[0068]Streptomyces coelicolor is an excellent model strain to studyantibiotic production and other process; it is the genetically moreintensively studied Streptomyces species and produces four chemicallydifferent antibiotics, whose biosynthetic genes have been isolated: theblue-pigmented polyketide actinorhodin, undecylprodigiosin,methylenomycin and the calcium-dependent antibiotic. Among these,actinorhodin is a representative secondary metabolite and possesses atypical polyketide biosynthetic pathway. Characters of actinorhodin:fine red needles from dioxane, dec. 270° C. Absorption max (dioxane):560, 523 nm. Soluble in piperidine, tetrahydrofuran, dioxane, phenol;slightly soluble in alcohol, acetic acid, acetone. Practically insol inaq acid; sol in aq alkali with bright blue color.

[0069] Characteristics of drugs:

[0070] Gentamicin:

[0071] Composition: this antibiotic complex consisting of threecomponents: GentamicinC₁, GentamicinC₂ and GentamicinC_(1a). As shown inFIG. 10, they possess similar structures.

[0072] Action mechanism: act on ribosomal 16S RNA or other ribosomalproteins (L6 protein or other unknown proteins) resulting in inhibitingprotein synthesis in ribosome.

[0073] Class: belonging to aminoglycoside antibiotics.

[0074] Geneticin:

[0075] Composition: single component and its structure as shown in FIG.10.

[0076] Action mechanism: inhibiting protein synthesis by acting on 16Sribosomal RNA or unknown proteins.

[0077] Class: belonging to aminoglycoside antibiotics.

[0078] Streptomycin:

[0079] Composition: single component and structure as shown in FIG. 10,which exists large difference with those of gentamicin and geneticin.

[0080] Action mechanism: inhibiting protein synthesis by acting onribosomal S12 protein and/or 16S RNA.

[0081] Class: belonging to aminoglycoside antibiotics.

[0082] Rifampicin:

[0083] Composition: single component and structure as shown in FIG. 10.

[0084] Action mechanism: inhibited RNA synthesis by acting on β-subunitof RNA polymerase.

[0085] Class: belonging to ansamycin antibiotics.

[0086] As an example of a preferred embodiment of this invention,actinorhodin productivity in Streptomyces coelicolor can be increased byintroducing each antibiotic resistant mutations into the strain at ahigh frequency of 5-10% among streptomycin-resistant,gentamicin-resistant or rifampicin-resistant isolates. Most mutants arestable and able to grow and form aerial mycelia normally as well as wildtype strain. Moreover, by introducing another antibiotic resistantmutation into single mutant, the actinorhodin productivity can befurther enhanced 1.8-2.2 fold. Of course, the level of increase maydiffer among the mutants. Finally, the third mutation can be introducedinto double mutants leading to triple mutants. Overproduction ofactinorhodin in the triple mutants is then tested. These antibioticspossess different action positions so that their functions are additiveand can be combined together for continuous improvement of antibioticproducers as shown in FIGS. 2, 4, 5 and 7. Mutational analyses showedthat most rifampicin resistant mutants produced a point mutation withinrpoB gene, encoding β-subunit of RNA polymerase, leading to highresistance levels to rifampicin; some streptomycin resistant mutantswith high resistance level to streptomycin revealed a point mutation inrpsL gene encoding ribosomal S12 protein, but no mutation in this genefor low level resistant mutants; all gentamicin or geneticin resistantmutants showed no mutation in rpsL gene and their mutations maybe existin an unknown gene. Although some mutational positions have not yet beendetermined, those mutations should exist in certain genes because theantibiotics used in this invention are known to act on the certaintargets in ribosome.

[0087] Random mutagenesis and selection is referred to as the class ofapproach or non-recombinant strain improvement procedure. Improvedmutants are normally identified by screening a large population ofmutated organisms, since the mutant phenotype may not be easy torecognize, the desired mutations occur at an extremely low frequency.Although this approach has the advantage of being simple and reliable,random screening is time-consuming and costly.

[0088] Moreover, random mutation screening using mutagens possesses nocertain targets and show a relatively large uncertainty. In contrast,method described in this invention use some antibiotics inhibitingprotein or nucleic acid synthesis as screening agents, which areentirely distinguishable from traditional mutagens such as radiationrays, chemicals (base analogs, deaminating agents, alkylating agents orintercalating agents etc.) and biological agents (phage, plasmid or DNAtransposons etc.). Moreover, the method described in this invention canproduce high frequency of desired mutation so that selection of positivemutants do not need much time and labor.

[0089] Multi-drug resistant microorganisms which are obtained in thepresent invention can be used as heterogous hosts to express foreigngenes encoding agriculturally and/or medically useful antibiotics,enzymes and biologically active substances, which are objective.

[0090] In addition to the manipulation of microorganisms by mutation,the techniques of genetic recombination provide another rational methodfor improving strains. It can be used to relieve rate-limiting steps ofthe biosynthetic pathway by increasing, for example, the gene dosage, oraltering a regulation mechanism. Obviously this requires much knowledgeof antibiotic production biochemistry and genetics like Streptomycescoelicolor and E. coli. However, most new antibiotic producers lack muchknowledge so that this method is not applicable for most strains.Moreover, this method is costly and complex. In contrast, the methoddescribed in this invention does not require much knowledge ofbiochemistry and genetics of strains, and is simple and not costly. Thisnew method can be used for the most microorganisms includingStreptomyces or other bacteria, especially for the wild type strainsisolated from nature.

[0091] In addition to improving productivity of strains, the methoddescribed in this invention can be used to activate certain silentantibiotic biosynthetic genes by introducing drug-resistant mutations.The present inventors isolated a number of Streptomyces sp. from soils,which show no antibacterial activity and then selected the antibioticresistant mutants with ability to produce antibacterial activity. Thepresent inventors found that about 50% strains isolated from soils canbe activated to produce antibiotics as shown in FIG. 9 and Table 3.Meanwhile, the antibiotic syntheses in mutants are medium-dependent asshown in FIG. 9. Therefore, it is important to use several differentkinds of media to examine the productivity of antibacterial agents.

EXAMPLES

[0092] Examples of the preferred embodiments of the present inventionwill be described herein below. It should be understood that the presentinvention is not limited to these examples.

Example 1

[0093] Spore stock of Streptomyces coelicolor wild type strain wasspread on the GYM agar containing 0.4% of glucose, 0.4% of yeastextract, 1% of malt extract, 0.1% of peptone (NZ-Amine, Type A), 0.2% ofNaCl and 2% of agar (before adding agar, adjust pH to 7.3). The mediumwas sterilized by an ordinary method (121° C., 15 min.). Then, the agarplates were incubated for 10-14 days at 30° C. to allow sporulation. Thesterile distilled water (5 ml) was added to each plate and the surfacescraped gently to release the spores. The suspensions were collected bycentrifugation and washed twice with distilled water. Before use forinoculum, the spores were dispersed for 10 min. in a sonic bath. Theconcentrations of spores were adjusted to about 2×10₉ spores per ml.

[0094] The spontaneous streptomycin, gentamicin or rifampicin-resistantmutants of S. coelicolor were obtained as the colonies that grew within7 days at 30° C. after spores (or cells) were spread on GYM agar(composition is the same as the above GYM agar) containing 5 μgstreptomycin per ml or 1.0 μg of gentamicin per ml or 200 μg ofrifampicin per ml, respectively (Growth of parental strain was inhibitedcompletely with 1 μg of streptomycin per ml, 0.1 μg of gentamicin per mlor 10 μg of rifampicin per ml). The single colonies grew on screeningplates were picked out and inoculated to R4 agar containing 1% ofglucose, 0.1% of yeast extract, 0.001% of Casamino Acids, 0.3% ofproline, 1% of MgCl₂·6H₂O, 0.4% of CaCl₂·2H₂O, 0.002% of K₂SO₄, 0.56% ofTES [N-Tris (hydroxymethyl)methyl-2-aminoethanesulfonic acid], 0.2% oftrace elements solution (as described for R2YE medium) and 2% of agar(before adding agar, adjusted to pH 7.2). The R4 plates were incubatedat 30° C. for 6 days and the amount of blue pigment actinorhodinproduced was compared among wild type strain and mutants, and selectedthe over-producers among the mutants. Then, the mutants withover-producing actinorhodin were inoculated into R4 liquid medium(composition is the same as the R4 agar, but no agar) in 25 ml of testtubes containing 5 ml medium on an orbital shaker at 30° C., 350 rpm.After incubation for 6 days, 1 ml culture samples were taken andadjusted pH to 8.0. After centrifugation at 1100 g for 5 min.,actinorhodin production in supernatants was assayed by measurement ofthe optical density of supernatants at 633 nm. The highest productivitydetected reached 1.6-fold for gentamicin-resistant mutants, 1.8-fold forstreptomycin-resistant mutants and 3.0-fold for rifampicin-resistantmutants. The frequencies for such high producers were 5%, 6% and 10%,respectively (see Table 1 and FIG. 7).

[0095] Mutational analyses of the mutants were carried out by theprocedure as described below. The rpsL gene fragment of the Strresistant mutant was obtained by PCR using a mutant genomic DNA as atemplate and synthetic oligonucleotide primers P1 (forward:5′-ATTCGGCACACAGAAAC) and P2 (reverse: 5′-AGAGGAGAACCGTAGAC) designed onthe basis of sequence of S. lividans (DDBJ accessionno: D83746). PCR wasperformed by following the manufacturer's instructions and using Taqpolymerase (Takara Ex Taq). A Perkin-Elmer Cetus thermal cycler wasused, and conditions were 5 min. of incubation at 96° C., followed by 30cycles of 96° C. for 0.3 min., 55° C. for 0.2 min. and 72° C. for 0.5min., finally at 72° C. for 10 min. PCR products were directly sequencedby the dideoxy chain-termination procedure using the Bigdye TerminatorCycle Sequencing kit (Perkin-Elmer Applied Biosystems, Foster City,Calif., U.S.A). The sequence data were analyzed with the GENETIX program(Software Development Co., Tokyo, Japan).

[0096] The partial rpoB gene fragment of the Rif resistant mutant wasobtained by PCR using a mutant genomic DNA as a template and syntheticoligonucleotide primers P3 (forward: 5′-GGCCGCTACAAGGTGAACAAGAAG) and P4(reverse: 5′-CGATGACGAAGCGGTCCTCC) designed on the basis of sequence ofS. coelicolor M145. The PCR method and DNA sequencing are the same asthose in zpsL gene. The rpsL genes of 3 gentamicin-resistant mutants and3 streptomycin-resistant mutants were sequenced and compared to that ofStreptomyces coelicolor wild type strain. The partial rpoB genes of 3rifampicin-resistant mutants were sequenced and compared to that ofStreptomyces coelicolor wild type strain. Three rifampicin-resistantmutants exhibited a point mutation in this region of rpoB gene; only onestreptomycin-resistant mutant possessed a point mutation within rpsLgene; All gentamicin-resistant mutants showed no mutation in rpsL gene(see Table 2).

Example 2

[0097] The spore solution of strain S-1 (a streptomycin-resistantmutant) was prepared by the same procedure as used in Example 1.

[0098] The spontaneous geneticin, gentamicin or rifampicin-resistantmutants of S-1 were obtained as colonies that grew on GYM agarcontaining 2.5 μg of geneticin per ml or 2.5 μg of gentamicin per ml or200 μg of rifampicin per ml. (Growth of S-1 strain was inhibitedcompletely with 0.5 μg of geneticin per ml, 0.5 μg of gentamicin per mlor 10 μg of rifampicin per ml.). Actinorhodin productivity of mutantswas examined using R4 agar plates and R4 liquid medium.

[0099] The highest productivity detected reached 1.7-fold forgeneticin-resistant mutants, 2.2-fold for gentamicin-resistant mutantsand 2.5-fold for rifampicin-resistant mutants. The frequencies of thesehigh producers were 13%, 14% and 18%, respectively (see Table 1 and FIG.7). Mutational analyses of the mutants were carried out by the procedureas described in Example 1. All three antibiotic resistant mutantsobtained here kept the mutation in rpsL gene from S-1, but no additionalmutation was found in rpsL gene. Seven rifampicin-resistant mutantsproduced a point mutation in rpoB gene (see Table 2).

Example 3

[0100] The spore solutions of SG-1 and SG-2 strains (gentamicin- andstreptomycin-resistant double mutants) were prepared by the sameprocedure as used in Example 1.

[0101] The spontaneous rifampicin-resistant mutants of SG-1 and SG-2strains were obtained as colonies that grew on GYM agar containing 200μg of rifampicin per ml. (Growth of SG-1 and SG-2 strains werecompletely inhibited with 10 μg of rifampicin per ml.) for 7 days at 30°C.

[0102] Actinorhodin productivity of mutants was examined using R4 agarplates and R4 liquid medium.

[0103] The highest productivity detected reached 3.4-fold forrifampicin-resistant mutants of SG-1 strain and 3.6-fold forrifampicin-resistant mutants of SG-2 strain. The frequencies of suchhigh producers were 10% and 15%, respectively (see Table 1 and FIG. 7).Mutational analyses were carried out by the procedure as described inExample 1. Five rifampicin-resistant mutants were found to have a pointmutation in rpoB gene, but one mutant had no mutation in this region(see Table 2). TABLE 1 Screening and antibiotic productivity ofdrug-resistant mutants Concn. of Frequency(%) of Highest Actinorhodinantibiotic used mutants producing productivity productivity MIC forscreening increased detected Strain (OD₆₃₃)^(a) (μg/ml)^(b) (μg/ml)actinorhodin^(c) (OD₆₃₃)^(d) S. coelicolor 0.77 Gentamicin (0.1) 1.0  5(4/80) 1.25 1147 Streptomycin(1.0) 5  6 (7/120) 1.39 (wild type)Rifampicin(10) 200 10 (15/150) 2.32 S-1 1.28 Geneticin (0.2) 2.5 13(15/112) 2.22 Gentamicin(0.1) 2.5 14 (14/104) 2.80 Rifampicin(10) 200 18(21/116) 3.14 SG-1 2.02 Rifampicin(10) 200 10 (8/80) 6.88 SG-2 1.88Rifampicin(10) 200 15 (12/80) 6.68

[0104] TABLE 2 Summary of mutations on the S. coelicolor rpsL or rpoBgene resulting in amino acid exchange Amino acid Amino acid ResistancePosition in position Position in position level (μg/ml)^(c) to: StrainrpsL gene^(a) (exchange) rpoB gene^(b) (exchange) STR^(d) GEN RIF GNE1147 —^(e) 1 0.1 10 0.2 S-1 262A→G 88(Lys→Glu) 100 S-2 ND^(f) 5 S-3 ND10 G-1 ND 0.3 G-2 ND 0.3 G-3 ND 0.3 R-1 1049G→A 350(Arg→His) 400 R-21040A→G 347(His→Arg) 400 R-3 1049G→T 350(Arg→Phe) 400 SGe-1 262A→G88(Lys→Glu) 100 0.5 SGe-2 262A→G 100 1 SGe-3 262A→G 100 1 SG-1 262A→G100 0.3 SG-2 262A→G 100 0.3 SG-3 262A→G 100 0.3 SR-1 262A→G 995T→C332(Leu→Arg) 50 50 SR-2 262A→G 1154C→T 385(Pro→Leu) 50 150 SR-3 262A→G1179C→G 393(Ile→Met) 50 400 SR-4 262A→G 1011C→A 337(Asp→Glu) 100 400SR-5 262A→G 1010A→G 337(Asp→Gly) 100 400 SR-6 262A→G 1028C→T343(Ser→Leu) 50 400 SR-7 262A→G 1048C→T 350(Arg→Cys) 50 400 SGR-1 262A→G1039C→T 347(His→Tyr) 100 0.2 400 SGR-2 262A→G 1041C→A 347(His→Gln) 1000.2 400 SGR-3 262A→G ND 50 0.3 300 SGR-4 262A→G 1039C→T 347(His→Tyr) 500.2 400 SGR-5 262A→G 1054A→T 352(Asn→Tyr) 50 0.5 400 SGR-6 262A→G1027C→T 343(Ser→Pro) 100 0.3 300

Example 4

[0105] The time courses of actinorhodin biosynthesis were carried out byusing Streptomyces coelicolor wild type strain, S-1 (astreptomycin-resistant mutant), SG-1 (a gentamicin and streptomycinresistant double mutant) and SGR-1 (a genetamicin-, streptomycin- andrifampicin-resistant triple mutant). Erlenmeyer flask of 500 ml-capacitycontaining 150 ml of R4 or R3 medium was inoculated with sporesolutions, then incubated at 30° C. on an orbital shaker at 200 rpm forthe denoted time. The composition of R4 liquid medium was the same asused in Example 1. The R3 liquid medium was the same as R4 but containedan increased amount (0.5%) of yeast extract and an extra KH₂PO₄(0.005%). At 24 h, 48 h, 72 h, 96 h, 120 h, 144 h or 168 h of incubationfor R4 medium (at 36 h, 60 h, 84 h, 108 h, 132 h, 156 h, 180 h ofincubation for R3 medium), 1 ml culture samples were taken and adjustedpH to 8.0. After centrifugation at 1100 g for 5 min., actinorhodinproduction was assayed by measurement of the optical density ofsupernatants at 633 nm. The results as shown in FIG. 5, these single,double and triple mutants displayed in hierarchical order a remarkableincrease in the actinorhodin biosyntheses.

Example 5

[0106] The effects of nutritional source on actinorhodin production wereinvestigated by using the strains and procedures as described in Example4. R4 liquid medium, was used as the basic medium, which wassupplemented with different amounts of yeast extract, Casamino acids orKH₂PO₄, respectively. Actinorhodin assay was the same as described inExample 4. As shown in FIG. 6, supplementation of yeast extract resultedin the severe impairment of actinorhodin productivity. This result wasless pronounced in the triple mutant. Unlike yeast extract, Casaminoacids was effective for enhancing actinorhodin production in the single,double or triple mutants but not the wild type strain, demonstrating theefficacy of those drug-resistant mutations. KH₂PO₄ had virtually noeffect on actinorhodin productivity.

Example 6

[0107] The unidentified Streptomyces isolated from soils were inoculatedto GYM agar for preparing spore solutions as described in Example 1. Thescreening of streptomycin, gentamicin or rifampicin resistant mutantswas as described in Example 2. The resistant mutants were inoculated toGYM agar, R4 agar and SYM agar, and incubated at 30° C. for 6 days. Thecomposition of GYM agar and R4 agar was the same as described inExample 1. SYM agar contained 1% of soluble starch, 0.2% of yeastextract, 0.5% of glucose and 2% agar (before adding agar, adjust pH to7.3).

[0108] The agar plug method was used to detect productivity of anantibiotic in mutants by measuring the extent of growth inhibition(diameters of inhibitory zones) of test organisms (E. coli K12, S.aureus 209P, B. subtilis 6633 or Candida albicans).

[0109] The results of screening are shown in FIG. 9 and Table 3. TABLE 3Results of screening for the strains from No. 101 to 200 Finishing date:May 09, 2000 Starting date: Feb. 06, 2000 100 Number of strains testedNo. 618749 to No. 618892 Number of strains producing 95 drug-resistantmutants Number of strains producing 45 mutants that produce antibioticsNumber of strains producing 6 the mutants to act on E. coli K12 Numberof strains producing 17 the mutants to act on C. albicans Number ofstrains producing the 38 mutants to act on S. aureus 209P Number ofstrains producing 39 the mutants to act on B. subtilis 6633

Example 7

[0110] A gentamicin-resistant mutant and a rifampicin-andgentamicin-resistant mutant of Bacillus cereus No. 2045 were producedaccording to a similar manner to that of Examples 1, 2 or 3. Thosemutants were precultured in bouillon medium for 10 h. Cells (0.1 ml)were inoculated into 5 ml of production medium consisting of (per liter)20 g of polypeptone, 20 g of corn steep liquor, and 5 g of NaCl(adjusted to pH 7.5 with NaOH) for 2 days at 30° C. The results areshown in Table 4. TABLE 4 Screening results and characteristics ofmutants Resistance Resistance Productivity*⁴ level to level to (μg/ml)of Strain Genotype GEN*² (μg/ml) RIF*² (μg/ml) WB2045*³ B. cereusWild-type 0.8 1.0 60 No. 2045 BG-1 gen*¹ 4.0 1.0 126 BG-2 gen 4.0 0.8135 BG-3 gen 4.5 1.0 130 BGR-1 gen-rif 4.5 100 380 BGR-2 gen-rif 5.0 120340 BGR-3 gen-rif 5.0 150 285 # contained (per liter) 17 g of nutrientbroth (Difco), 17 g of glucose, 3.4 g of NaCl, 8.5 mg of CuSO₄.5H₂O,12.75 mg of FeSO₄.7H₂O, 6.12 mg of MnSO₄.5H₂O, 25.5 mg of CaCl₂.2H₂O,and 15.3 mg of ZnSO₄.7H₂O (adjusted to pH 7.2 with NaOH)] for 48 hr at30° C.

Example 8

[0111] A rifampicin-resistant mutant and a streptomycin-andrifampicin-resistant mutant of Streptomyces lividans 66 were producedaccording to a similar manner to that of Examples 1, 2 or 3, and theactinorhodin production was estimated. The results are shown in Table 5.TABLE 5 Screening results and characteristics of mutants ResistanceResistance Productivity level to RIF level to STR of Act strain genotype(μg/ml)*¹ (μg/ml)*¹ (OD₆₃₃ nm)*² S. lividans 66 Wild-type 10 1.0 0.089SR-1 rif¹ 200 1.0 0.265 SR-2 rif 400 0.8 0.864 SR-3 rif 400 1.0 0.684SRS-1 rif-str 400 50 1.200 SRS-2 rif-str 350 120 1.801 SRS-3 rif-str 400100 1.342

[0112] While the invention has been described in detail and withreference to specific embodiments thereof, it will be apparent to oneskilled in the art that various changes and modifications can be madetherein without departing from the spirit and scope thereof.

[0113] This application is based on U.S. Provisional Patent ApplicationNo. 60/279,665 filed Mar. 30, 2001, the entire contents thereof beinghereby incorporated by reference.

INDUSTRIAL APPLICABILITY

[0114] The present invention provides a method for increasing theproductivity of the secondary metabolite by a microorganism in alabor-saving, time-saving, high efficient and semi-rational way andbeing applicable for the strains without more knowledge of, for example,antibiotic biochemistry and genetics.

1 5 1 17 DNA Artificial Sequence synthetic oligonucleotide 1 attcggcacacagaaac 17 2 17 DNA Artificial Sequence synthetic oligonucleotide 2agaggagaac cgtagac 17 3 24 DNA Artificial Sequence syntheticoligonucleotide 3 ggccgctaca aggtgaacaa gaag 24 4 20 DNA ArtificialSequence synthetic oligonucleotide 4 cgatgacgaa gcggtcctcc 20 5 65 PRTStreptomyces coelicolor 5 Gln Leu Ser Gln Phe Met Asp Gln Asn Asn ProLeu Ser Gly Leu Thr 1 5 10 15 His Lys Arg Arg Leu Asn Ala Leu Gly ProGly Gly Leu Ser Arg Glu 20 25 30 Arg Ala Gly Phe Glu Val Arg Asp Val HisPro Ser His Tyr Gly Arg 35 40 45 Met Cys Pro Ile Glu Thr Pro Glu Gly ProAsn Ile Gly Leu Ile Gly 50 55 60 Ser 65

1. A method for increasing a productivity of a secondary metabolite in amicroorganism by conferring a resistance against two or more antibioticsto said microorganism.
 2. A method for obtaining a microorganism havingan increased productivity of a secondary metabolite, which comprises thesteps of conferring a resistance against two or more antibiotics to saidmicroorganism by culturing it in a medium containing the antibiotics,wherein the concentration of said antibiotics is higher than MIC of saidantibiotics against the original microorganism, and isolating colonieswhich can grow in the medium.
 3. The method as claimed in claim 1 or 2,wherein said microorganism is a bacterium.
 4. The method as claimed inclaim 1 or 2, wherein said microorganism belongs to the genus selectedfrom the group consisting of Streptomyces, genus Bacillus, and genusPseudomonas.
 5. The method as claimed in claim 1 or 2, wherein saidmicroorganism is selected from the group consisting of Streptoymcescoelicolor, Streptoyces lividans, Streptomyces antibioticus,Streptomyces chattanoogensis, Bacillus subtilis, Bacillus cereus, andPseudomonas pyrrocinia.
 6. The method as claimed in claim 1 or 2,wherein said antibiotic is selected from the group consisting ofribosomal protein-attacking antibiotics, ribosomal RNA-attackingantibiotics, and RNA polymerase-attacking antibiotics.
 7. The method asclaimed in claim 1 or 2, wherein said antibiotic is selected from thegroup consisting of streptomycin, geneticin, gentamicin, and rifampicin.8. The method as claimed in claim 1 or 2, wherein said secondarymetabolite is selected from the group consisting of antibiotics, enzymesand physiologically active substances.
 9. A method for producing asecondary metabolite, which comprises the steps of culturing in a mediuma microorganism having a resistance against at least two antibiotics andhaving an increased productivity of the secondary metabolite incomparison to the original microorganism thereof, forming andaccumulating a secondary metabolite, and recovering the secondarymetabolite therefrom.
 10. The method as claimed in claim 9, wherein saidmicroorganism is a bacterium.
 11. The method as claimed in claim 9,wherein said microorganism belongs to the genus selected from the groupconsisting of Streptomyces, genus Bacillus, and genus Pseudomonas. 12.The method as claimed in claim 9, wherein said microorganism is selectedfrom the group consisting of Streptomyces coelicolor, Streptomyceslividans, Streptomyces antibioticus, Streptomyces chattanoogensis,Bacillus subtilis, Bacillus cereus, and Pseudomonas pyrrocinia.
 13. Themethod as claimed in claim 9, wherein said antibiotic is selected fromthe group consisting of ribosomal protein-attacking antibiotic,ribosomal RNA-attacking antibiotics, and RNA polymerase-attackingantibiotics.
 14. The method as claimed in claim 9, wherein saidantibiotic is selected from the group consisting of streptomycin,geneticin, gentamicin, and rifampicin.
 15. The method as claimed inclaim 9, wherein said secondary metabolite is selected from the groupconsisting of antibiotics, enzymes and physiologically activesubstances.
 16. A microorganism having an increased productivity of thesecondary metabolite in comparison to an original strain thereof, whichis produced in accordance with claim 1 or
 2. 17. The microorganism asclaimed in claim 16, wherein said microorganism is a bacterium.
 18. Themicroorganism as claimed in claim 16, wherein said microorganism belongsto the genus selected from the group consisting of Streptomyces, genusBacillus, and genus Pseudomonas.
 19. The microorganism as claimed inclaim 16, wherein said microorganism is selected from the groupconsisting of Streptomyces coelicolor, Streptomyces lividans,Streptomyces antibioticus, Streptomyces chattanoogensis, Bacillussubtilis, Bacillus cereus, and Pseudomonas pyrrocinia.
 20. A method forincreasing a productivity of a secondary metabolite in a microorganismby conferring a resistance against a single antibiotic, exceptingstreptomycin, to said microorganism.